bioRxiv preprint doi: https://doi.org/10.1101/828426; this version posted November 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. N-cadherin SPRY motifs bind unconventionally-secreted Fbxo45 and regulate multipolar neuron migration Abbreviated title: N-cadherin, Fbxo45 and multipolar neuron migration Youn Na1,3, Elif Kon2, Hong Cao1,3, Yves Jossin2,4, Jonathan A. Cooper1,4 1 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98019, USA 2 Laboratory of Mammalian Development & Cell Biology, Institute of Neuroscience, Université catholique de Louvain, 1200 Brussels, Belgium 3 Present Addresses: YN; Samsung NeuroLogica, Danvers, MA HC; Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, 200032, China 4 Contributing authors Correspondence: Email: [email protected] Number of pages: 40 Number of Figures: 8; Supplementary Tables 3; Supplementary Figures 5 1 bioRxiv preprint doi: https://doi.org/10.1101/828426; this version posted November 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. ABSTRACT During development of the mammalian neocortex, the orientation of migrating multipolar projection neurons is controlled by Reelin, a secreted glycoprotein, which increases cell-surface expression of N-cadherin. Although N-cadherin regulates cell-cell adhesion, recent results suggest that its adhesive function is not required to orient multipolar neuron migration. To understand N-cadherin function in multipolar migration, we performed two independent screens for embryonic brain proteins that bind the N-cadherin extracellular domain. Both screens detected MycBP2 and SPRY-domain protein Fbxo45, two components of an intracellular E3 ubiquitin ligase. We found that Fbxo45 is secreted by a non-classical mechanism, not involving a signal peptide and not requiring endoplasmic reticulum to Golgi transport. Secreted Fbxo45 stimulated neurite branching in culture. A SPRY-motif N-cadherin mutant did not bind Fbxo45 and failed to rescue neuron migration even though it still formed trans-homophilic adhesions. The results suggest that secreted Fbxo45 may regulate neurite branching and bind N-cadherin to orient multipolar neuron migration. 2 bioRxiv preprint doi: https://doi.org/10.1101/828426; this version posted November 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. INTRODUCTION The complex architecture of the mammalian neocortex arises through the generation, specification, migration and connection of different types of neurons (1-3). Projection neurons, born and specified in the pallial ventricular zone (VZ), migrate outwards to form discrete layers before they establish connections. Their migrations pass through discrete stages. They first migrate radially from the VZ to the subventricular zone (sVZ)/intermediate zone (IZ), where they become multipolar and migrate randomly (4). When they arrive in the upper IZ, they assume a bipolar morphology and migrate radially outwards, passing by the earlier-born neurons in the cortical plate (CP) until they stop at the marginal zone (MZ) and undergo terminal somal translocation. Genetic disruption of pathways that regulate neuron migration is associated with neurodevelopmental disorders including lissencephaly, epilepsy and schizophrenia. Classical cadherins, including neuronal (N-)cadherin (NCad) and epithelial (E-)cadherin (ECad), are calcium-dependent cell-cell adhesion molecules (5). NCad is important for cell-cell adhesion in the neuroepithelium of the VZ (6, 7). Somewhat surprisingly, NCad also regulates projection neuron migration at two stages: first to orient multipolar neurons towards the CP (8- 10), and second, for bipolar neurons to undergo terminal translocation (11-13). NCad is upregulated on the cell surface at both these stages in response to an extracellular signal, Reelin. Reelin is known to regulate neuron migration and cortical development by stimulating signaling pathways involving Src kinases, Dab1, PI3’ kinase, Crk/CrkL, C3G, and the small GTPase, Rap1 (14-21). Rap1 then regulates NCad surface expression, presumably through Rab GTPase-dependent vesicular trafficking events (9, 11, 22). However, the downstream events by which NCad regulates multipolar neuron migration have not been elucidated. Because cadherin trans-interactions are critical for cell-cell adhesion and recognition (5), we hypothesized that homophilic interactions between NCad on migrating neurons and NCad on other neurons, axons, or radial glia may orient multipolar migration. Indeed, neurons in culture will polarize towards an external source of NCad (23). However, our recent work 3 bioRxiv preprint doi: https://doi.org/10.1101/828426; this version posted November 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. revealed that a NCadW161A mutant, which cannot form “strand-swap” trans homodimers (24-27), stimulates multipolar migration (28). We further found that NCad binds and activates fibroblast growth factor receptors (FGFRs) in cis (on the same cell) and this interaction is needed for multipolar migration (28). ECad is not able to replace NCad for this function even though binds FGFR. Mechanistically, NCad but not ECad protected FGFR from being degraded, and the first two of five extracellular calcium-binding domains on NCad (EC1 and EC2) were critical. These results leave open the possibility that additional proteins binding NCad EC1 or EC2 may regulate neuron migration. Fbxo45 (F box/SPRY domain-containing protein 1) is a little-studied protein that is highly expressed in the nervous system and required for cortical lamination, axonal outgrowth and synaptic connectivity (29-31). Most F-box proteins bind Skp1, Cul1 and Rbx1 to form a SCF (Skp1-Cul1-F-box) E3 ubiquitin ligase complex. Fbxo45 is atypical in that it does not bind Cul1 or Rbx1 and instead associates with MycBP2/PAM (Myc binding protein 2/protein associated with Myc), forming a Fbxo45-Skp1-MycBP2 complex that has E3 ligase activity in vitro (30). The SPRY domain of Fbxo45 potentially interacts with substrates. Curiously, NCad was detected in a Fbxo45 interaction screen (32). Furthermore, knockdown of Fbxo45 decreased NCad expression and impaired differentiation of neuronal stem cells (32), suggesting that Fbxo45 interaction with NCad is involved in brain development. Here we set out to identify secreted proteins that interact with the ectodomain of NCad and may regulate radial migration. Two different unbiased proteomics approaches detected Fbxo45 and MycBP2 as major binding partners for the extracellular domain of NCad. We found that the SPRY domain of Fbxo45 binds a SPRY domain consensus in the EC1 region of NCad but not ECad. Furthermore, Fbxo45 is secreted by an unconventional mechanism from neurons and other cells and regulates dendritic arborization in vitro. Cell autonomous knockdown of Fbxo45 interfered with neurogenesis, which precluded analysis of neuron migration. Therefore, we tested the potential role of Fbxo45 in neuron migration by an alternative approach. We 4 bioRxiv preprint doi: https://doi.org/10.1101/828426; this version posted November 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. generated a NCad mutant which does not bind to Fbxo45 but still forms calcium-regulated trans- homophilic interactions. Unlike NCadW161A, which binds Fbxo45 and rescues multipolar migration (28), the mutant NCad that does not bind Fbxo45 did not rescue multipolar migration. These results suggest that secreted Fbxo45 may stimulate NCad-dependent neuron migration during brain development. RESULTS BioID identifies Fbxo45 and MycBP2 as extracellular NCad-interacting neuronal proteins. We used two approaches to identify NCad binding proteins. First, we adapted proximity- dependent biotin identification (BioIDf) for extracellular use (33). BioID uses the BirA mutant (R118G, BirA*) to generate reactive biotinyl-AMP from biotin and ATP. Biotinyl-AMP rapidly reacts with nucleophiles in the immediate surroundings, including ε-amino groups of lysine residues on nearby proteins (33). BioID has been used to detect binding partners of relatively insoluble proteins including those at cell junction complexes (34-37). The working distance of BirA* is ~10 nm (34), which is about half the length spanned by the five EC repeats that comprise the NCad ectodomain (38) (diagrammed in Figure 1A). To detect proteins that might interact with either end of the NCad ectodomain, we inserted Myc-BirA* into HA-tagged NCad (NCad-HA) either between EC5 and the transmembrane domain (TM) (N5-BirA*)